1. Field of the Invention
The present invention relates to an optical biosensor using an immunological reaction, a fluorescent marker and a detection principle based on measuring fluorescence and using an evanescent light wave to excite and sense fluorescence.
2. Description of the Prior Art
Conventionally, a biosensor of this type is an optical system comprising a waveguide to which light is coupled with a wavelength specific to a fluorescent agent that can be excited by the evanescent wave in the optical waveguide.
The immunological reaction employed in a biosensor of this type can essentially be brought about by two different methods.
In a first method, antibodies specific to an antigen to be detected are fixed on the surface of the waveguide. The sample to be analyzed, whose antigen concentration is to be ascertained, is mixed with antigens that are identical to those which are to be detected but are chemically bound to a fluorescent agent (also referred to as a "marker" or "label").
This hybrid sample is then incubated and the fluorescence excited by the evanescent wave is measured. Under these conditions, if the sample contains a high quantity of antigens (in which case it is referred to as "positive"), few antigens bound to the fluorescent agent will couple to the antibodies immobilized on the surface of the waveguide, and so weak fluorescence will be measured. However, if the sample initially contains few antigens or no antigens at all (in which case it is referred to as "negative"), those which have been added with the fluorescent agent will bind to the immobilized antibodies and strong fluorescence will be measured.
In a second method, the procedure adopted is somewhat reversed. In this case, antigens of the same type as those to be detected are bound beforehand to a protein and are immobilized on the surface of the waveguide. The sample is mixed with antibodies which are specific to this antigen but are bound to a fluorescent agent. This sample is brought into contact with the antigens immobilized beforehand on the surface of the waveguide and the fluorescence is measured after incubation.
In this case, if the sample is positive the antibodies provided with the fluorescent agent will bind to the antigens of the sample instead of binding to the antigens immobilized on the surface of the waveguide. The antigen/antibody/fluorescent-agent complexes will then remain remote from the surface of the waveguide and weak fluorescence will be measured.
However, if the sample is negative the antibodies provided with the fluorescent agent will bind to the antigens immobilized on the surface of the waveguide. The fluorescent agent can then be excited by the evanescent wave and strong fluorescence will be measured.
The measurement may be qualitative, and also quantitative if the intensity of the observed fluorescence is measured.
The waveguide may be formed by a solid substance which conducts light and on whose surface diffraction gratings may be provided for coupling into it an excitation beam originating from a light source and for extracting from it output radiation due to the fluorescence and the original radiation not converted by the fluorescence. Since the radiation due to the fluorescence has a wavelength different to that of the incident radiation, they are separated angularly by the grating and a photodetector may be placed in the beam of the fluorescence, the output signal of this photodetector being representative of the quantity of antigens present in the sample.
Measurement with a biosensor of this type suffers from interference inherent to its structure or the ambient conditions, for example the quality of coupling/decoupling of the light beams, losses of light energy in the waveguide and the effects of temperature.
In order to take an absolute measurement, it is therefore necessary to provide reference zones on the waveguide, whose fluorescence is known and which are provided in the sensor in the same way as the measurement zones, as described for example in U.S. Pat. No. 5,631,170. A reference zone is also required because the biosensor is preferably disposable and measurement conditions may therefore differ from one biosensor to another because of unavoidable manufacturing tolerances, especially pertaining to the dimensions and the surface condition of the waveguide of the biosensor.
However, these reference zones have a number of drawbacks. First, they take up space on the surface of the waveguide, thereby reducing the space available for the measurement zones.
Further, the reference zones are necessarily provided at positions other than those where the measurement zones are located and where the measurement conditions are not identical (different surface conditions, different waveguide thicknesses, etc.). The biosensor therefore has an inherent inaccuracy, in spite of the reference zones and the secondary measurement which they involve.
The aim of the invention is to provide an optical biosensor using an immunological reaction in which the measurement reference can be obtained simply and reliably without reducing the area available for the measurement, which is independent of the dimensional inaccuracy of a mass-produced biosensor.